Cell surface profiling of cultured cells by direct hydrazide capture of oxidized glycoproteins

Glycoproteins are a particularly interesting subset of the cellular proteome as a high proportion of proteins present on the extracellular cell surface are glycosylated. These cell surface proteins are ideal targets for biologic drug therapies or for diagnostics tests. Here, we describe a modification of the well-described Cell Surface Capture (CSC) method for the selective isolation and identification of cell surface glycoproteins that contain N-linked carbohydrates. This modification, which we refer to as Direct Cell Surface Capture (D-CSC), is based on oxidation of cell surface glycans on intact cells, followed by direct conjugation of the oxidized oligosaccharides to a solid support using hydrazide chemistry, with no biotinylation step. As a proof-of-principle, we applied D-CSC to the analysis of cell surface membrane proteins of three adherent cancer cell lines (A549, OVCAR3, and U87MG) and compared our results to those published using the well-established Cell Surface Capture (CSC) method, demonstrating comparable selectivity for cell surface proteins. • A method enabling the identification of cell surface proteins from cells in culture is described.• Application of this method to profile the cell surface on three different cancer cell lines is included.

aldehyde and a hydrazide group on a gel-based bead.Glycoproteins immobilized on the bead are subsequently digested with trypsin or chymotrypsin and then peptides containing an N-linked glycosylation site are released using PNGaseF.These N-glycopeptides are then analyzed by mass spectrometry to identify cell surface proteins.The original published CSC method [8] also oxidizes glycans on the cell surface, but then reacts the resulting aldehydes with a modified biotin (biocytin hydrazide) molecule.Proteins are then digested with trypsin and biotinylated peptides are immobilized on streptavidin resin prior to release with PNGaseF.While the biotinylation step appears to be added to improve the cell surface selectivity of modification, our results suggest that direct capture of oxidized glycoproteins is equally selective for cell surface proteins.

Glycan oxidation of cell surface proteins
Material and equipment required for 2 × 150 mm plates of adherent cells: -Phosphate-buffer saline (PBS), pH 6.5 (adjusted if needed) and chilled to 4 °C -Oxidation solution: 10 mM Sodium meta periodate (NaIO 4 ) ○ Place 0.086 g of NaIO 4 in a 50 mL conical tube covered with aluminum foil.○ Immediately prior to use, add 40 mL of cold PBS pH 6.5.-10X Quenching solution: 200 mM Sodium Sulfite (Na 2 SO 3 ) ○ Place 0.252 g of Na 2 SO 3 in a 15 mL conical tube.○ Immediately prior to use, add 10 mL of cold PBS pH 6.5.-Large container filled with crushed ice in which 2 plates can fit side by side -15 and 50 mL plastic conical tubes -Aluminum foil -Timer -1 mL pipette and tips -Cell scraper -Refrigerated swinging-bucket centrifuge capable of centrifuging 15 and 50 mL conical tubes.
Note: Typically, 20-30 million cells are adequate for good results.We often use 2 large (150 mm) plates to obtain > 1 mg of total protein for adherent cell lines.However, the number of plates might need to be increased depending on cell type.2. For adherent cell lines, place the cell culture plates on a flat bed of crushed ice to inhibit cellular trafficking of cell membrane proteins.For suspension cell lines, gently pellet the cells in a 50 mL conical tube by centrifugation at 200 g for 5 min at 4 °C and place the tube on ice.Note: This protocol describes the cell surface labeling of adherent cell lines while they are still attached to the plate.This method has the advantage of maintaining high cell viability and integrity, but may bias surface profiling results to the exposed apical surface.If desired, a soft detachment method suitable to the adherent cell line can be used at this stage to put adherent cells into suspension while maintaining cell viability.Detached adherent cells can be treated as a suspension cell line for the remainder of this protocol.Detachment may result in increased labeling of the basal cell surface and increased identification of basal surface proteins.Detachment may lead to loss of some surface receptors, especially if an enzymatic detachment method is used, and some contamination with intracellular ER or Golgi-resident glycoproteins may occur if membrane integrity is disrupted during detachment.3. To expose the cells to 10 mM NaIO 4 , carefully remove the cell media and slowly add 18 mL of cold Oxidation solution to each plate/tube.4. Protect from light by covering with foil and incubate for 10 min on ice rocking plates every 2 min to ensure the cells do not dry out.It is generally not necessary to rock tubes. 5. Stop the oxidation reaction by adding 1900 μL of cold 10X Quenching solution to each plate/tube for a final concentration of ∼20 mM Na 2 SO 3 .6. Mix gently by rocking each plate/tube a few times and then incubate for an additional 6 min on ice.Rock plates every 2 min to ensure the cells do not dry out.7.For suspension cells, skip to Step 8.For adherent cells on plates: a. Transfer 8 mL of the liquid from each plate into a single 50 mL plastic tube placed on ice.This solution may contain some detached cells.b.Using a cell scraper, scrape the cells into the remaining buffer and transfer those cells into the same 50 mL tube.c.Add an additional 5 mL of cold PBS pH 6.5 to each plate.Swirl to re-suspend any remaining cells then combine the cell suspension into the 50 mL conical tube as well.8. Pellet the cells by centrifugation at 400 g for 5 min at 4 °C, then carefully remove the supernatant.
Note: Centrifugation speed and time should be adjusted as needed for each cell line.9. Re -suspend the cells in 10 mL cold PBS, pH 6.5 and transfer into a 15 mL tube.10.Pellet the cells by centrifugation at 400 g for 5 min at 4 °C.11.Remove PBS and proceed directly with cell lysis or freeze cell pellet at − 80 °C.

Cell Lysis
Material and equipment: -Hydrazide (Hz) Coupling buffer: 100 mM NaOAc, 150 mM NaCl, pH 5.  Protease digest and washes : 25.After removing the last wash, plug the bottom of the column.26.Add 1:100 w/w of sequencing grade trypsin ( i.e. add 10 μg trypsin for processing 1 mg of cell lysate) diluted in 500 μL of Digest Solution or add 1:100 of chymotrypsin diluted in 500 μL of 50 mM AMBIC to the Hz beads.Note: Using both trypsin and chymotrypsin in separate digestion of the same sample is optional, but generally improves the number of identifications.If only a single protease is being used, trypsin is the best choice.27.Cap the top of the column and apply parafilm around the plug and also the cap to prevent leakage.28.Invert a few times to re-suspend the Hz beads and place the spin column in a labeled 1.5 mL tube.29.Place the sample in the head over head tube rotator and use parafilm again to secure the sample on the tube holder of the rotator.30.Place the tube rotator in the incubator set at either 37 °C for trypsin digestion or at 25 °C for chymotrypsin digestion for an O/N digestion (12 to 18 h).31.Place the column into a new 2 mL tube and centrifuge at 100 g for 15 s.
Note: The flow through contains the protease-released peptides which can be frozen at − 80 °C and analyzed by Mass Spec separately.However, we find that this fraction is often not cell surface specific.32.Transfer the column in a new 2 mL tube and remove the remaining non-glycosylated peptides with a series of washes with salts and organic solvents using a Vacuum manifold or by centrifugation as described previously.33.Wash the Hz beads 3 times with 500 μL of 1.5 M NaCl and then 3 times with 500 μL of Organic Wash Solution (60% ACN-0.1% TFA) and 3 times with 500 μL of MeOH.34.Finally, wash the Hz beads 6 times with 500 μL of 50 mM AMBIC to completely remove the organic solvents.
Release of N-linked glycopeptides with PNGase F: 35.Using a widened tip, add 500 μL of 50 mM AMBIC to the Hz beads and transfer them from the spin column into a new labelled 1.5 mL tube, if necessary.36.Spin the tube for 1 min at 1500 g and then very carefully pipet out the AMBIC buffer without disturbing the bed of Hz beads.37. Add 100 μL of 50 mM AMBIC containing 4 units of PNGase F to the Hz beads.
Note: 4 units of PNGase F is typically used when processing an input of 1 mg of cell lysate.However, we've noticed some variability in the activity (units/mg) of PNGase F from lot to lot.If the number of N-linked peptide are lower than expected OR if the amount of intact protein (PNGase F) is high in the sample leading to contamination of the LC system, the amount of PNGase F should be adjusted accordingly.38.Gently flick the tube to re-suspend the Hz beads and then incubate O/N (12 to 18 h) at 37 °C without rotation.
Note: Since the sample volume is small, there is no need for rotation.39.Transfer the supernatant that now contains the released N-linked glycopeptides to a new 1.5 tube.40.To maximize peptide recovery, add 100 μL more of 50 mM AMBIC to the Hz beads, flick the tube a few times to re-suspend the beads, let the beads settle then pool the supernatant with the previously collected N-linked glycopeptides.41.Repeat the previous step one more time.42.Pre-wet a new mini-spin column with 400 μL of 50 mM AMBIC by centrifuging it for 1 min at 100 g inside a 2 mL tube.43.Remove any leftover Hz bead particles from the released N-linked glycopeptides fraction by spinning the sample through the pre-wetted column into a new 1.5 mL tube for 1 min at 100 g.

Note:
We analyze the N-linked fraction without any additional clean-up step.If a C18 cleanup step (e.g.stage tip) is incorporated prior to mass spectrometry analysis, this step may not be required.44.Speed vac the sample down to concentrate the peptides.
Note: for an input of 1 mg of oxidized cell lysate, the sample is typically brought down to a volume of about 30 to 60 μL.45.Freeze the sample at − 80 °C until ready to analyze.

Notes on mass spectrometry (MS) and data analysis:
While this protocol is focused on the sample preparation side for cell surface proteomics, we have identified some important considerations when doing MS analysis of these samples, as outlined below: • In our experience, only the fraction of peptides released by PNGaseF are specific for cell surface proteins.The peptides released by trypsin or chymotrypsin are highly enriched in cell surface proteins, but also contain many non-membrane proteins due to non-specific binding.• It is important to include deamidation of asparagine as a variable modification during the database search to identify peptides that have had a glycan removed by PNGaseF.• For maximum specificity for cell surface proteins, it is important to filter peptide identifications to require the presence of a deamidated N-linked glycosylation consensus site (N-X-S/T/C where X is any amino acid except P) when producing the final peptide identification list.• When using chymotrypsin, a less specific peptide search is required.It is important to take special care with managing falsediscovery rates, as these can be inflated with the more open database search.• In general, analysis of cell surface peptides is improved by MS acquisition methods that are focused on sensitivity of detection, rather than speed of MS2 acquisition.
of cell surface proteins as defined by SURFY [1] or Sobotski et al. [5] .The original CSC method identified a higher percentage of cell surface proteins as defined by the human Cell Surface Protein Atlas (CSPA).This finding was not unexpected since the CSC analysis of U87MG cells analyzed here was one of the datasets used to build the CSPA.Taken together, these results suggest that we did not lose significant cell surface specificity by eliminating the biotinylation step and that it is possible that cell surface selectivity was improved when using D-CSC.D-CSC has some minor advantages over the original CSC protocol.It is often practical for the cell surface labeling step to be done by collaborators who are experts in growing the cells of interest.In D-CSC, these collaborators can simply oxidize the cells as outlined in the "Glycan oxidation of cell surface proteins " section, freeze the cell pellet, and ship it to the mass spectrometry lab for sample processing.The glycan oxidation steps in D-CSC require only inexpensive reagents that can be easily purchased by the collaborating lab.It uses methods that are very familiar to any scientist with tissue culture expertise and does not require chemical labeling with a specialized biotin reagent like CSC.The second advantage of D-CSC is that it enables analysis of the protease-released fraction.While this fraction is not specific to the cell-surface, it is enriched in cell surface proteins and can provide interesting protein identifications in comparative proteomic experiments that are not seen in whole cell peptide digests or in the cell surface profiling.This would include surface glycoproteins with only o-linked glycosylation or that have N-linked glycosylation sites that fall in peptides that are not amenable to MS identification.In CSC, the protease-released fraction is generally contaminated with abundant streptavidin peptides which can limit loading and lead to contamination of nano-LC systems, while in D -CSC this fraction can be more easily analyzed with optimal loading to increase identifications.

3 .
Centrifuge for 10 min at RT at 4500 g to pellet any cell debris.4.Transfer the cleared cell lysate into a new 15 mL conical tube. 5. Quantify protein content of the cleared cell lysate using the Detergent Compatible (DC) protein assay (Bio-Rad).6. Adjust cell lysate to a protein concentration between 1 and 1.3 mg/mL by diluting sample with additional Lysis Buffer.7. Proceed directly with the hydrazide capture of oxidized glycoproteins, or aliquot and freeze the cell lysate at − 80 °C until ready to proceed.Hydrazide (Hz) capture of oxidized glycoproteinsMaterial and equipment:-Affi-Gel R ○ Hz Hydrazide Gel (Biorad, Cat# 153-6047)-Hydrazide (Hz) Coupling buffer: 100 mM NaOAc, 150 mM NaCl, pH 5.5) -Urea Buffer: 8 M Urea, 0.4 M ammonium bicarbonate ○ Place 19.2 g of Urea and 0.6 g of ammonium bicarbonate in 50 mL conical tube and add H 2 O until volume reaches the 40 mL mark.-50 mM AMBIC: 50 mM Ammonium Bicarbonate ○ Dissolve 0.16 g of ammonium bicarbonate in 40 mL of dH 2 O. -10 mM Dithiothreitol (DTT) ○ Add 0.015 g of DTT in a 15 mL conical tube and add 10 mL of 50 mM AMBIC -25 mM Iodoacetamide (IAA) ○ Add 0.046 g of IAA in a 15 mL conical tube and add 10 mL of 50 mM AMBIC -Trypsin Digestion Solution: 50 mM AMBIC -15% Acetonitrile (ACN) ○ Pipette 6 mL of Acetonitrile into a 50 mL plastic tube.○ Bring volume up to the 40 mL mark with dH 2 O. ○ Add 0.16 g of Ammonium Bicarbonate.-Acetonitrile (ACN) -1.5 M NaCl ○ Dissolve 43.83 g of NaCl into 500 mL of H 2 O. ○ Keep at RT for up to 1 year.-Organic Wash Solution: 60% Acetonitrile (ACN)-0.1% Trifluoroacetic acid (TFA) ○ Mix 8 mL of ACN with 12 mL of H 2 O. ○ Add 20 μL of TFA.-100% Methanol (MeOH) -Sequencing grade Trypsin (Promega, Cat# V5113) and/or Chymotrypsin (Sigma, Cat# C3142) -PNGase F (Sigma, Cat# F8435) -1.5 and 2 mL tubes -Pierce centrifuge columns 0.8 mL (Thermo Fisher Scientific, Cat# 89,868), caps and end caps (Bio-Rad, #731-1660) -Pipettes and tips -Gel loading tip (200 μL) -Parafilm -MACSMix head-over-head shaker (Miltenyi Biotec) or a similar tube rotator -Benchtop centrifuge -Heat bloc and/or incubator -Speed Vac concentrator -Vacuum manifold (optional) Procedure: Preparation of Affi-Gel R ○ Hz Hydrazide Gel (Hz beads) : 5)○ Add 6.8 g of sodium acetate trihydrate (C 2 H 9 NaO 5 ) and 4.4 g of NaCl into 450 mL of H 2 O. ○ Adjust pH to 5.5 with acetic acid ○ Adjust volume to 500 mL with H2O ○ Sterilize with 0.22 filter unit.○ Store at room temperature (RT) for up to 1 year.-Lysis Buffer: 0.5% SDS in Hz coupling buffer with 1/200 of protease inhibitor cocktail (Sigma, cat# P8340) and 1/1000 benzonase (Sigma, Cat# 71,205-3) ○ Add 0.025 g of SDS in 5 mL of Hz Coupling buffer in a 15 mL conical tube.○ Immediately prior to use, add 1/200 of protease inhibitor cocktail and 1/1000 benzonase into the required volume of Add 1 to 5 mL of Lysis Buffer to the cell pellet to lyse the cells.Homogenize the cells by passing the sample through a 3 mL syringe 5 times.Place sample tube the rotator at RT for 30 min to 2 h until all the DNA has been degraded by the benzonase (until the sample is no longer viscous).
Note: The volume of Lysis Buffer is adjusted according to the pellet size.Use a volume approximately equivalent to 10X of the pellet size (i.e. for a cell pellet with a volume around 200 μL, use 2 mL of Lysis Buffer.2. Note: Use a syringe with a 33 gage needle to completely aspirate and eject the cell lysate 5X.It is also possible to sonicate at this step to break up the DNA, possibly eliminating the need for benzonase treatment.However, we have found it difficult to control foaming of the SDS-based buffer during sonication.